Method of detecting cancer using delta-catenin

ABSTRACT

The present invention provides a method for detecting or screening for the presence of cancer in a subject. The method comprises obtaining, providing or collecting a tissue or fluid sample (such as a urine sample) from said subject, and then determining the presence or absence of delta-catenin in said sample, or increased levels of delta-catenin in said sample as compared to a normal or control subject. The presence of delta-catenin in said sample, or increased levels of delta-catenin in said sample, indicating said subject is afflicted with or at least at risk of developing cancer.

RELATED APPLICATION DATA

This application is a continuation application of and claims priority toU.S. application Ser. No. 13/251,481, filed Oct. 3, 2011, now U.S. Pat.No. 8,932,824, which is a continuation application of U.S. applicationSer. No. 12/238,539, filed Sep. 26, 2008, now U.S. Pat. No. 8,058,020,which is a continuation application of U.S. application Ser. No.10/872,692, filed Jun. 21, 2004, now U.S. Pat. No. 7,445,906, whichclaims priority to U.S. Provisional Patent Application Ser. No.60/483,666, filed Jun. 30, 2003, the disclosures of each application areincorporated herein by reference in their entireties.

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A sequence listing in ASCII text format, submitted under 37 C.F.R.§1.821, entitled 5218-109TSCT3_ST25.txt, 737 bytes in size, generated onMar. 23, 2015, and filed electronically via EFS-Web, is provided in lieuof a paper copy. The Sequence Listing is incorporated herein byreference into the specification for its disclosures.

BACKGROUND OF THE INVENTION

Prostate cancer is a major cause of death among men in Westerncountries. The current protocol for detection of this cancer involvestesting for prostate-specific antigen (PSA) levels. If PSA levels arefound to be high (4 ng/ml), a tissue biopsy is performed. Unfortunately,PSA testing is limited by the fact that it lacks sensitivity and it doesnot distinguish between prostate cancer and benign prostate hyperplasia.As a result, many men either are not identified as having the disease orbecause of false positive tests are subjected to the invasive tissuebiopsies when they do not have the disease. A much more specific andless invasive diagnostic test is needed for early detection of thisdisease.

Delta-catenin presents itself as an improved alternative to thePSA/biopsy tests currently utilized for prostate cancer detection. Deltacatenin was first identified and patented (U.S. Pat. No. 6,258,929) as aneurospecific protein, alternatively named ALARM. At the time, theprotein was believed to be expressed almost exclusively in brain tissue.However, Burger, et al. (Int. J. Cancer 100, 228-237 (2002))subsequently found the messenger RNA for delta-catenin to be expressedin prostate cancer tumors with the delta-catenin transcripts beinglocalized to the glandular secretory cells. Unlike PSA, delta-cateninwas capable of distinguishing between prostate cancer and benignprostate hyperplasia. Burger et al. noted a possible diagnostic role fordelta-catenin in prostate cancer detection. However, they also pointedout that a significant difficulty remained in development of this toolsince delta-catenin had only been detected in glandular secretoryepithelial cells in prostate tissues and had not been found in prostatestroma or bodily fluids, such as serum or urine.

SUMMARY OF THE INVENTION

The present invention shows that delta-catenin mRNA and protein areexpressed not only in prostate cancer tissues but also in many othercancer types. For example, in esophageal cancer, delta-cateninexpression, like in prostate cancer, increases with the increasing tumorgrade. Thus, its usefulness may extend beyond detection of prostatecancer. Moreover, it has now been found that when over-expressed,delta-catenin can be detected not only in the affected epithelialtissues but also in the extracellular spaces and stroma. As a result,the invention has overcome the barrier in using delta-catenin as aspecific, non-invasive diagnostic tool for prostate and other types ofcancer.

Thus the present invention provides a method for detecting or screeningfor the presence of cancer in a subject. In general, the methodcomprises obtaining, providing or collecting a tissue or fluid sample(such as a urine sample) from said subject, and then determining thepresence or absence of delta-catenin in said sample, or increased levelsof delta-catenin in said sample as compared to a normal or controlsubject. The presence of delta-catentin in said sample, or increasedlevels of delta-ctanin in said sample, indicating said subject isafflicted with or at least at risk of developing cancer.

A further aspect of the present invention is a method as describedabove, wherein the presence of at least one additional cancer biomarkersuch as a cadherin, prostate specific antigen, and/or p120 in thesubject is also detected. The presence of at least one additionalbiomarker indicates the subject is more likely afflicted with cancer;the absence of at least one additional biomarker indicates the subjectis less likely afflicted with cancer. The use of multiple biomarkers, atleast one of which is delta-cadherin as described herein, serves toreduce the number of false positives and false negatives detected by anyone biomarker alone, particularly where the other biomarker is acadherin such as E-cadherin or p120.

A further aspect of the present invention is the use of ananti-delta-catenin antibody for carrying out a method as describedabove.

Another aspect of the present invention is the use of a nucleic acidamplification technique such as the polymerase chain reaction (PCR),particularly reverse transcriptase-polymerase chain reaction (RT-PCR)with delta-catenin specific primers for carrying out a method asdescribed above.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationset forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Expression of delta-catenin protein in cancer and itsassociation with cancer progression.

Panel A. A schematic drawing illustrating the full length structure ofthe delta-catenin protein, consisting of 1247 amino acids. MAbD30, UB1,RAb64, RAb08, and RAb07 are epitopes to which antibodies have been made.No clear significance in protein function by these epitopes is known,but it is believed that rAb64 contains the armadillo domain that may beinvolved in protein-protein interaction; rAb08 contains several proteinphosphorylation sites that may be important for signal transduction;rAb08 also contains a PDZ binding motif that directs binding to PDZproteins. PDZ proteins are a diverse group of scaffolding proteins thatare important to bring proteins together for function.

Panel B. Expression of delta-catenin in prostate cancer (CaP). 40 ug ofproteins were loaded in each lane. Lane 1 is the D30 epitope, lane 2 isthe UBI epitope, etc.).

FIG. 2. Western blot demonstrating differential expression ofdelta-catenin in various tissues as compared to an actin control.

Panels A and B. Each lane was loaded with 40 μg protein from fourdifferent types of tissue culture cells. Lane 1, cortical neurons; Lane2, PZ-HPV-7 non-cancerous human prostate cells; Lane 3, HS-5 human bonemarrow stromal cells; Lane 4, CWR-R1 human prostate tumor cells. Panel Awas immunoblotted with anti-delta catenin antibodies. Panel B wasimmunoblotted with anti-actin antibodies.

Panels C and D. Each lane was loaded with 80 μg of protein from eitherCWR22 xenograft tumors (Lanes 1-3) or mouse brain tissue (Lane 4). Lane1, tissue taken at initial tumor formation; Lane 2, 2 dayspost-castration; Lane 3, 150 days post-castration; Lane 4, mouse braintissue. Molecular weight is indicated on the left. Panel C wasimmunoblotted with anti-delta catenin antibodies and Panel D wasimmunoblotted with anti-actin antibodies. (Note that castration willreduce androgen level in the body and cause the tumor to shrink;delta-catenin expression is down 2 days after castration, whichcoincided with tumor shrinkage and cell apoptosis/cell death, but wasback up after 150 days post-castration, a condition called tumorrecurrence that is androgen independent).

FIG. 3. A transmission electron micrograph showing excretion ofdelta-catenin into extracellular spaces by cells in which the protein isbeing over-expressed. Arrows indicate the structures reacting withdelta-catenin antibody.

FIG. 4. Evidence of promotion of cell invasion by delta-catenin.

Panel A. Fluorescent light microscope photographs of prostate tumorcells. Insets A and B: prostate tumor cells that have welldifferentiated morphology show strong staining for p120ctn (A), but notdelta-catenin; Insets C and D: prostate tumor cells that show moremotile phenotype display strong delta-catenin staining, but a reducedp120ctn staining; Insert: higher magnification showing high expressionof delta-catenin as clusters in Inset D.

Panel B. A graphic presentation of the percent cell of invasion promotedby delta-catenin as compared to a control. Lamin B dsRNA, delta-catenindsRNA, delta-catenin cDNA. A BD Bioscience MATRIGEL™ Invasion Chamberassay was used. The chamber assay apparatus consists of two chambers.The upper chamber has a cursion of matrix protein called Matrigel andhas cells plated on top of that. Invasive cells will be able topenetrate the gel and migrate to the lower chamber. More cells on thelower side of the chamber indicates higher potential for invasion.

FIG. 5. Delta-catenin expression in prostate, breast, ovarian,pancreatic, colon, adrenal cancer and leukemia cells. Different cancertypes were probed with delta-catenin antibody or an actin antibodycontrol. Lane 1, endothelial cells; Lane 2, HS-5, human bone marrowstromal cell line; Lane 3 PZ-HPV-7, normal human prostate epithelialcells; Lane 4, CWR-R1, prostate cancer cell line; Lane 5, MCF-7, breastcancer cells, Lane 6, Jurkat, human leukemia T cell line, Lane 7,Ovarian cancer cells; Lane 8, Panc-1, human pancreatic cancer cell line;Lane 9, HT-29, human colon adenocarcinoma cells; Lane 10, PC12-δ-cat,pheochromocytoma cells (adrenal cancer) (δ-cat=transformed withdelta-catenin). This particular figure shows the transformed PC12 cells,in which delta-catenin band is used as positive control fordelta-catenin).

FIG. 6. Tissue Microarray showing a high percentage of prostate cancercases with upregulated delta-catenin expression. The samples come fromcommercial source of prostate cancer tissues, are well characterizedprostate cancer samples (meaning that the tumor grade and Gleason scoresare known, and are immunostained with anti-delta-catenin).

FIG. 7. Delta-catenin overexpression in the extracellular spaces ofprostate cancer tissue. Arrows point to glandular secretory epithelialcells, whereas arrowheads point to extracellular matrix and stroma).

Panel A. Normal prostate tissue shows glandular structure withoutdelta-catenin immunoreactivity.

Panel B. Stage 1 tumor shows glandular structure with minimaldelta-catenin immunoreactivity.

Panel C. Stage 2 tumor shows enhanced delta-catenin immunoreactivity inthe secretory epithelial cells of the gland without extracellularstaining.

Panel D. Stage 4 tumor shows strong delta-catenin immunoreactivity inthe extracellular space.

FIG. 8. δ-Catenin overexpression in primary human prostaticadenocarcinomas.

Panel A. Semi-quantitative immunoscore showing low immunoreactivity ofδ-catenin (mean+s.e. of immunoscore=2.52+0.04) in benign samples andhigh immunoreactivity of δ-catenin (mean+s.e. of immunoscore=7.58+0.05)in prostatic adenocarcinomas. An immunoscore was obtained as: extentscore×intensity score (range, 0 to 12).

Panel B. Semi-quantitative immunoscore showing increases in meanimmunoscore with prognostically increased Gleason score. The immunoscorewas obtained similarly as in Panel A.

FIG. 9. Increased δ-catenin immunoreactivity is accompanied by thereduced E-cadherin and p120^(ctn) immunostaining during prostate cancerprogression. A and B. Correlation between the changes in the intensity(Panel A) and extent (Panel B) of δ-catenin immunoreactivity and theGleason scored prostatic adenocarcinomas. Panels C and D. Correlationbetween the changes in the intensity (Panel C) and extent (Panel D) ofE-cadherin immunoreactivity and the Gleason scored prostaticadenocarcinomas. E and F. Correlation between the changes in theintensity (Panel E) and extent (Panel F) of p120^(ctn) immunoreactivityand the Gleason scored prostatic adenocarcinomas. Asterisks: p<0.05

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

“Cancer” as used here includes but is not limited to brain, lung,breast, colon, prostate, esophageal, ovarian, pancreatic, adrenal, skincancer and leukemia cells.

“Subject” as used herein refers to animal subjects, particularlymammalian subjects, including but not limited to, humans, non-humanprimates, dogs, cats, rabbits, goats, horses, pigs, and cattle. Thesubject may be a male subject or a female subject and may be of all agesincluding infant, juvenile, adolescent and adult subject.

“Fluid sample” or “body fluid sample” as used herein includes but is notlimited to blood samples, seminal fluid, urine, and fine needleaspirates from suspected afflicted organs such as the prostate in asubject.

“Blood sample” as used here refers to whole blood, blood plasma, bloodserum or any fraction thereof, so long as the fraction contains (insubject with cancer) delta-catenin as described herein.

“Antibody” as used herein refers to intact immunoglobulin moleculeshaving binding affinity to delta-catenin, as well as immunologicallyactive fragments thereof, such as Fab, Fab′, F(ab′)2, etc. Antibodiesinclude any type of immunoglobulin and may be monoclonal, polyclonal orchimeric and may be of any species of origin, including (for example)mouse, rat, rabbit, horse, or human. See, e.g., M. Walker et al. Molec.Immunol. 26, 403-11 (1989), may be substituted or unsubstituted, and maybe naturally occurring or synthetic.

The disclosures of all U.S. patent references cited herein are to beincorporated by reference herein in their entirety.

The present invention is, as noted above, drawn to methods for detectionof cancer utilizing delta-catenin. Detection may be for diagnostic orprognostic purposes, may be for general screening purposes, may be fortargeting cancer in chemotherapy, or may be for the purpose ofdetermining if a subject is at risk of developing cancer, confirm adiagnosis, indicate the reoccurrence of cancer, etc.

One aspect of the invention is drawn to obtaining a fluid sample from asubject and determining the presence or absence of delta-catenin in saidfluid sample; the presence of delta-catenin indicating that said subjectis afflicted with or at least at risk for developing cancer. In onepreferred embodiment, the fluid sample is a blood sample.

As noted above, the subject may be a human subject, or an animal subjectfor veterinary or drug screening or development purposes, with examplesof suitable animal subjects including but not limited to dogs, cats,rabbits, goats, horses, pigs, cattle, etc. The subjects may be a malesubject or a female subject; the subject may be of any age includinginfant, juvenile, adolescent and adult subjects.

The present invention may be used to detect any type of cancer,including but not limited to esophageal, lung, breast, colon, ovarian,pancreatic, adrenal, skin cancer or leukemia. In one preferredembodiment of the invention, the cancer to be detected is prostatecancer.

As noted above, the present invention provides a method of screening forthe presence of cancer in a subject, comprising the steps of: (a)contacting a biological sample taken from said subject with an antibodyor delta-catenin specific primers as described above under conditionspermitting said antibody or primers to specifically bind an antigen ordelta-catenin nucleic acid sequences in the sample to form anantibody-antigen complex or PCR reaction complex; and then (b)determining the amount of antibody-antigen complex or delta-catenin RNAin the sample as a measure of the amount of antigen or RNA in thesample, wherein an elevated level of the antigen or RNA in the sample isassociated with the presence of cancer in said subject. Other techniquesfor determining the quantity of delta-catenin in a subject or sampletaken from a subject that do not involve antibodies and immunoassays orRT-PCR can also be used (e.g., chromatography techniques), but antibodyand RT-PCR assay techniques are currently preferred.

The delta-catenin (also called “ALARM”) protein and antibodies theretoare known. See, e.g., U.S. Pat. No. 6,258,929 to Kosik et al; Q. Lu etal., J. Neurosci Res. 67, 618 (2002), and antibodies for carrying outthe present invention can be produced in accordance with knowntechniques. For example, monoclonal antibodies of the present inventionmay be prepared using any technique which provides for the production ofantibody molecules by continuous cell lines in culture. These include,but are not limited to, the hybridoma technique, the human B-cellhybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al.(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030;Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120). Briefly, theprocedure is as follows: an animal is immunized with antigen orimmunogenic fragments or conjugates thereof. For example, haptenicoligopeptides of antigen can be conjugated to a carrier protein to beused as an immunogen. Lymphoid cells (e.g. splenic lymphocytes) are thenobtained from the immunized animal and fused with immortalizing cells(e.g. myeloma or heteromyeloma) to produce hybrid cells. The hybridcells are screened to identify those which produce the desired antibody.Polyclonal antibodies used to carry out the present invention may beproduced by immunizing a suitable animal (e.g., rabbit, goat, etc.) withthe delta-catenin antigen, collecting immune serum from the animal, andseparating the polyclonal antibodies from the immune serum, inaccordance with known procedures. Depending on the host species, variousadjuvants may be used to increase immunological response. Such adjuvantsinclude, but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

The step of determining the delta-catenin content of the fluid sample(for example, urine) may comprise contacting the urine sample withanti-delta catenin antibodies and measuring the amount of the anti-deltacatenin-antigen complex, wherein elevated levels of antigen-antibodycomplex indicates the presence of cancer. Any suitable assay format canbe used, a variety of which will be known to persons skilled in the art.

Those skilled in the art will be familiar with numerous specificimmunoassay formats and variations thereof which may be useful forcarrying out the method disclosed herein. See generally E. Maggio,Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see alsoU.S. Pat. No. 4,727,022 to Skold et al. titled “Methods for ModulatingLigand-Receptor Interactions and their Application,” U.S. Pat. No.4,659,678 to Forrest et al. titled “Immunoassay of Antigens,” U.S. Pat.No. 4,376,110 to David et al., titled “Immunometric Assays UsingMonoclonal Antibodies,” U.S. Pat. No. 4,275,149 to Litman et al., titled“Macromolecular Environment Control in Specific Receptor Assays,” U.S.Pat. No. 4,233,402 to Maggio et al., titled “Reagents and MethodEmploying Channeling,” and U.S. Pat. No. 4,230,767 to Boguslaski et al.,titled “Heterogenous Specific Binding Assay Employing a Coenzyme asLabel.”

Antibodies as described herein may be coupled or conjugated to a solidsupport suitable for a diagnostic assay (e.g., beads, plates, slides orwells formed from materials such as latex or polystyrene) in accordancewith known techniques, such as precipitation. Antibodies as describedherein may likewise be conjugated to detectable groups such asradiolabels (e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradishperoxidase, alkaline phosphatase), fluorescent labels (e.g.,fluorescein), chemiluminescent labels (e.g., acridinium groups,metalloporphyrins such as phthalocyanine dyes, luminol, etc.), metalatoms (e.g., technetium-99m), etc., in accordance with known techniques.See, e.g., U.S. Pat. No. 4,472,509 to Gansow (metal chelates tomonoclonal antibodies); U.S. Pat. No. 5,061,641 to Schochat et al.; andU.S. Pat. No. 4,861,869 to Nicoleotti et al. (radiolabelling proteins).

Amplification of nucleic acids may be carried out by any suitabletechnique, including but not limited to polymerase chain reaction(including, for RNA amplification, reverse-transcriptase polymerasechain reaction), ligase chain reaction, strand displacementamplification, transcription-based amplification (see D. Kwoh et al.,Proc. Natl. Acad. Sci. USA 86, 1173-1177 (1989)), self-sustainedsequence replication (or “3SR”) (see J. Guatelli et al., Proc. Natl.Acad. Sci. USA 87, 1874-1878 (1990)), the Q.beta. replicase system (seeP. Lizardi et al., Biotechnology 6, 1197-1202 (1988)), nucleic acidsequence-based amplification (or “NASBA”) (see R. Lewis, GeneticEngineering News 12 (9), 1 (1992)), the repair chain reaction (or “RCR”)(see R. Lewis, supra), and boomerang DNA amplification (or “BDA”) (seeR. Lewis, supra). The bases incorporated into the amplification productmay be natural or modified bases (modified before or afteramplification), and the bases may be selected to optimize subsequentelectrochemical detection steps. Techniques for amplification are knownand described in, among other things, U.S. Pat. Nos. 4,683,195;4,683,202; 4,800,159; and 4,965,188; G. Walker et al., Proc. Natl. Acad.Sci. USA 89, 392-396 (1992); G. Walker et al., Nucleic Acids Res. 20,1691-1696 (1992); R. Weiss, Science 254, 1292 (1991).

Immunoassays, or other types of assays to detect and/or quantitate thelevel of delta-catenin in samples as described below, may be used inscreening assays to detect pathologic states associated with aberrantlevels of delta-catenin expression (e.g., tumors, inflammatory states),diagnostic studies, prognostic studies, or to monitor the progression ordiminution of delta-catenin expression in correlation with diseasestate.

Samples that may be collected for use in carrying out the immunoassay ornucleic acid assay may be blood samples or tissue samples from the organor tissue of interest within the subject, such tissue generally of mostinterest being those types of tissues/cells that express differingamounts of delta-catenin in pathologic states as compared tonon-pathologic states, or biological fluids such as blood (includingblood fractions such as blood plasma or blood serum), urine,cerebrospinal fluid, etc). Examples may include overexpression oraberrant expression of delta-catenin in various types of malignancies asdescribed herein,

A biological sample may be a cell sample, with an intervening culturingstep being performed between the time the cell sample is collected fromthe subject and the immunoassay is carried out on the biological sample.

A biological sample may also be a cell, cell debris, or stroma sample,with an intervening step being performed before the sample is collectedfrom the subject to enhance the sensitivities of immunoassay.

For immunohistological techniques, a tissue sample is collected from thesubject, and the presence or absence of binding of an antibody of theinvention is detected. The presence of binding of the antibody in anabnormal pattern or a pattern indicative of a tumor or cancer indicatesthe presence of a tumor or cancer in the subject from which the tissuesample is collected. The presence of the antigen in a metastatic tumordeposit can also be used to determine a likely source of the primarytumor. Any suitable immunohistology format may be used. The tissuesample may include patient biopsies, resections or cells for cytologicstudy. A similar technique to immunohistology is the use of similartechniques to detect and/or phenotype cells in body fluids or othersuspensions as is used for flow cytometric examination.

For in vivo diagnostic purposes the antibody according to the inventionis coupled to or provided with a suitable externally detectable label,such as e.g. a radiolabel as described above or a metal atom (e.g.,technetium-99m), and administered to a subject (e.g., by intravenous orintraarterial injection), in an amount sufficient to produce anexternally detectable signal, whereupon the possible localizedaccumulation of antibody in the body is determined, with a localizedaccumulation of the antibody (in a region other than that which wouldordinarily be expected for normal subjects or subjects free of disease)indicating the present of a tumor in that subject.

Anti-delta-catenin antibodies can be packaged in any suitable form alongwith instructions for carrying out the methods described herein andprovided as a kit for carrying out the methods described herein, inaccordance with known techniques.

Another aspect of the invention involves the use of a combination ofbiomarkers to reduce the frequency of false positives or false negativesby the use of any one biomarker alone. For example, where a subject istested for δ-catenin in the manner described herein, that subject mayalso be tested for the presence of another biomarker. For example, thepresence of at least two biomarkers indicates that the subject is morelikely to be afflicted with cancer than if only one biomarker is foundin that subject; the absence of at least two biomarkers indicates thesubject is more likely to be free of cancer than if only one biomarkeris found in that subject; the presence of one biomarker in a subjectindicates the subject is more likely to be afflicted with cancer than ifthe subject is found to be free of another, different, biomarker, etc.Particular biomarkers that may be used in combination with the methodsof testing for δ-catenin as described herein include tight junction andadherens junction proteins such as claudin and cadherin (particularlyE-cadherin), prostate specific antigen, and p120 (particularlyp120c^(ctn)).

The presence or absence of other cancer biomarkers may be detected inaccordance with known techniques. Methods of detecting, diagnosing orscreening for cancer (particularly prostate cancer) by detecting thepresence of prostate specific antigen (PSA) are known and described in,among other things, U.S. Pat. Nos. 5,614,372; 5,840,501; 6,300,088;6,361,955; 6,383,759; 6,423,503; and 6,482,599. Methods of detecting,diagnosing or screening for cancer (including prostate cancer) bydetecting cadherins such as E-, OB-, N- and T-cadherin (particularlyE-cadherin) are known and described in, among other things, U.S. Pat.Nos. 5,597,725; 5,811,518; 5,997,866; 6,682,901; and 6,723,320. Methodsof detecting, diagnosing or screening for cancer by detecting p120(including p120^(ctn)) are known and described in, among other things,U.S. Pat. No. 4,902,615. The disclosures of all patent references citedherein are to be incorporated by reference herein in their entirety.

The present invention is illustrated in greater detail in the followingnon-limiting Examples.

Experimental

Antibodies specific for delta-catenin were developed using standardtechniques known in the art.

FIG. 1, Panel A identifies the specific epitopes of the delta-cateninprotein to which the antibodies were made. Expression of delta-cateninin prostate cancer cells is shown in FIG. 1, Panel B with the each ofantibodies reacting with the prostate cancer cells. FIG. 2, Panel Afurther demonstrates the differential expression of delta-catenin wherethe antibodies are shown to react specifically with cancerous prostatecells (lane 4) and not with non-cancerous prostate cells (lane 2) orbone marrow stromal cells. Additionally, as was previously known, thedelta-catenin is expressed in brain tissues (FIG. 2, Panel A, lane 1 andFIG. 2, Panel C, lane 4). FIG. 2, Panel B and 2, Panel D serve aspositive controls of constitutive levels of expression and also as acontrol to demonstrate a constant level of protein loading. Theexpression of delta-catenin in the cancerous but not in thenon-cancerous prostate cells demonstrates the potential improvement ofthis protein as a method of detecting prostate cancer over the commonlyused, less sensitive PSA test.

FIG. 6 shows upregulation of delta-catenin in a high percentage ofprostate cancer cases. The tissue array has many prostate cancer needlebiopsy samples spotted onto slides and immunostained withanti-delta-catenin. Among these prostate cancer samples each case wasidentified with certain tumor grade and Gleason score to indicate thetumor stage from early, more differentiated tumor tissues to late,poorly differentiated tumor tissue that may also be very invasive.Delta-catenin overexpression was seen in many of these samples and thehigher the tumor grade it is, the stronger the staining.

This invention further demonstrates the expression of delta-catenin in awide variety of cancer types in addition to prostate cancer (FIG. 5).Lanes 4-10 of FIG. 5, Panel A show delta-catenin expression in prostate,breast, ovarian, pancreatic, colon, adrenal cancer and leukemia cells.Delta-catenin antibody did not react with endothelial cells or bonemarrow stromal cells demonstrating its differential expression in onlyspecific tissue types (Lanes 1 and 2). FIG. 5, Panel B is anactin-antibody control to verify a constant level of protein loading.Low levels of delta-catenin were detected in prostate non-cancerouscells. So it is the level of protein rather than its presence or absencethat is of greatest importance. In general, the level of delta-cateninin normal tissue is very low, except for brain; In this case, the cellline PZ-HPV-7 is a non-cancerous cells, but to make it immortal and cangrow in culture, it is transformed with HPV). The tissue IHC, though,shows clearly that delta-catenin staining in normal prostate tissue isextremely low.

This invention for the first time demonstrates delta-catenin expressionin extracellular spaces (FIGS. 3 and 7). The transmission electronmicrograph (FIG. 3) of cells overexpressing delta-catenin showsextracellular structures that are immunoreactive with delta-cateninantibody. The progression of expression of delta-catenin expression indifferent stage prostate tumors is shown in FIG. 7. Normal prostateglandular tissue showed no immunoreactivity with delta-cateninantibodies (FIG. 7, Panel A). Stage 1 and stage 2 prostate tumor tissues(FIG. 7, Panel B and 7, Panel C) showed minimal to enhancedintracellular levels of delta-catenin immunoreactivity, respectively,while stage 4 tumor tissue (FIG. 7, Panel D) showed strong delta-cateninimmunoreactivity in the extracellular spaces. Extracellular expressionof delta-catenin is an important aspect in the development ofdelta-catenin as a non-invasive test for prostate cancer because it willallow the sampling of bodily fluids and not just tissues.

Experimental 2

Materials.

Mouse anti-E-cadherin, mouse anti-pp120 and mouse anti-δ-catenin D30 (toamino acids 85-194) were from BD Biosciences (Palo Alto, Calif.).Affinity purified rabbit anti-human δ-catenin was developed aspreviously described (Lu, Q., et al., J Cell Biol. 144 (3): 519-532,1999; Ho, C. et al., J Comp Neurol. 420 (2): 261-276, 2000). They wereraised against amino acids 434-530 (rAB62), amino acids 828-1022(rAb64), and amino acids 1213-1225 (rAb25). Mouse anti-actin was fromOncogene science (Boston, Mass.). Unless otherwise indicated, allchemicals were from Sigma (St. Louis, Mo.).

Cell Culture and cDNA Transfection.

PC12 cells were grown in DMEM with 10% heat inactivated horse serum and5% fetal bovine serum (FBS). Human endothelial cell lysates wereobtained from BD Biosciences (Palo Alto, Calif.). Non-tumorigenicprostate epithelial PZ-HPV-7 (ATCC, Rockville, Md.) was cultured inserum-free karotinocyte medium, while tumorigenic prostate cancer CWR-R1was cultured in Richter's improved minimal essential medium (Invitrogen,Carlsbad, Calif.) supplemented with epidermal growth factor (EGF),insulin/transferin/selenium, nicotinamide, linoleic acid, and FBS, asdescribed by Wu and Terrian (J Biol Chem. 277(43): 40449-40455, 2002).Primary hippocampal cultures were prepared as described by Goslin andBanker (1998) with minor modifications (Lu, Q. et al., J Cell Biol. 144(3): 519-532, 1999; Jones, S. B. et al., Neuroscience 115 (4):1009-1021, 2002). Briefly, 18-day timed pregnant rats were sacrificed,and the embryos were removed in accordance with the NIH Guide for theCare and Use of Laboratory Animals. Hippocampi and cortices werecollected, and cells were dissociated by trypsinization and plated ontopoly-L-lysine coated coverslips and cell culture plates, respectively.After neurons adhered to the substrate, the medium was changed over toB-27 supplemented Neurobasal. All cultures were maintained at 37° C.with 5% CO₂ until they were either fixed or lysed for immunochemicalanalysis.

Some cells were transfected with δ-catenin cDNA to evaluate the effectsof δ-catenin overexpression on p120^(ctn) expression and distribution.For neuronal cultures, hippocampal neurons grown for 5 days in vitro(DIV) were transfected with pEGFP (Clontech, Palo Alto, Calif.) ascontrol, or with pEGFP-δ-catenin using Lipofectamine 2000 reagent (LifeTechnologies Grand Island, N.Y.) according to instructions provided.Transfection of PZ-HPV-7 and CWR-R1 cells were performed with usingLipofectinamine-Plus method.

Protein Extraction and Immunoblotting.

Cultured cells were lysed in a radioimmunoprecipitation assay (RIPA)buffer containing 10 mM HEPES, pH 7.3, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 0.5% deoxycholate, 0.2% SDS with protease inhibitor cocktails.Insoluble materials were removed by centrifugation. For human benign andprostate tumor tissues, cell lysates were prepared from the frozenperipheral zones of prostatic acinar tissues by extraction in 150 mMNaCl, 50 mM Tris, pH 7.5, with 0.5% NP-40% with 1 mM sodium vanadate, 1mM Na Fluoride, 1 mM PMSF, 10 ug/ml leupeptin, 10 ug/ml pepstatin, and10 ug/ml aprotinin. The lysates were mixed with sample buffer and equalprotein amounts were loaded for SDS-PAGE analysis. After proteins weretransferred to nitrocellulose membranes (PGC Scientifics, Frederick,Md.), anti-δ-catenin (1:500 for rabbit antibodies and 1:300 for mouseantibodies) and anti-actin (1:5000) immunoreactivities were revealed byantibody immunoblotting using enhanced chemiluminescence (Amersham LifeScience, Piscataway, N.J.) for detection. When necessary, blots werestripped with 100 mM β-mercaptoethanol, 2% SUS, and 62.5 mM Tris-HCl, pH7.6 for 30 minutes at 55° C. The blots were then washed in Tris buffercontaining 0.1% Tween 20 before re-probing with different antibodies.

RNA Isolation and RT-PCR.

The total RNA was isolated by guanidinium thiocyanate-phenol-chloroformextraction (Jeansonne, B. et al., Cell Mol Biol. 49 (1): 13-21, 2003).To synthesize the first-strand DNA sequence from the total RNA, randomdecamers were used as a first-strand primer (Ambion Inc, Austin, Tex.).The specific PCR primers chosen to yield the 283 bp fragment ofδ-catenin are as follows: forward (5′-3′), TACTCCGCAAGACGACTGACC (SEQ IDNO:1), and reverse (5′-3′), CCATCACACTCTCTCATCCTTCTG (SEQ ID NO:2). Theannealing temperature in the PCR reaction is 56° C. Some of the RT-PCRproducts were purified from the DNA gel and verified by the DNAsequencing to confirm the δ-catenin sequence.

Tissue MicroArray Specimens.

The Tissue MicroArray (TMA) block was constructed from archival paraffinblocks of radical prostatectomy (RP) cases performed at VanderbiltUniversity Medical Center from January 1998 to December 2001. RPspecimens were submitted in their entirety for whole mount processing.Total tumor volume by digital planimetry, Gleason score, tumor stage,and status of surgical margins were assessed as described (Brooks, J. D.Microarray analysis in prostate cancer research. Curr Opin Urol. 12 (5):395-399, 2002). Ninety cases were retrieved from a maintained RPPathology database to achieve the following grade and stagecombinations: 15 Gleason score 5 or 6, pT2; 15 Gleason score 5 or 6,pT3; 15 Gleason score 7, pT2; 15 Gleason score 7, pT3; 15 Gleasonscore≧8, pT2; 15 Gleason score≧8, pT3 (Chung, C. H. et al., Nat Genet.32 Suppl: 533-540 (2002); Feroze-Merzoug, F. et al., Cancer MetastasisRev. 20 (3-4): 165-171 (2001)). Portions of tumor representative of thecase were identified by review of tumor maps and individual whole mountslides. Representative areas of tumor and benign (typically peripheralzone), corresponding to the zone of origin of the prostate cancer in thevast majority of cases (Brooks, supra), were identified on whole mountslides and matched to the corresponding paraffin blocks. The TMA blockwas constructed with a Beecher Instruments Tissue Array (Silver Spring,Md.) utilizing two representative 0.6 mm areas of tumor and one 0.6 mmarea of benign per case (total of 270 samples).

Immunohistochemistry.

For immunohistochemistry, 5 μM sections were cut from the TMA block ontocharged glass slides. The TMA slides were subjected to antigen retrievalfor all antibodies. After deparaffination and rehydration, tissuesections were placed in a pressure cooker on high for 15 minutes inTrilogy retrieval solution (Cell Marque, Hot Springs, Ark.). Sectionswere then immunostained with rabbit anti-δ-catenin, mouseanti-E-cadherin or anti-p120^(ctn) using Dako Autostainer with the LSAB2 protocol (labeled streptavidin-biotin method, Dako Corp, Carpenteria,Calif.). The streptavidin-biotin reaction was detected usingdiamionobenzidine as a substrate. All sections were counterstained withHematoxilin and coversliped using a Sakura Tissue Tek (Sakura Corp,Torrance, Calif.). TMA immunostaining was assessed in benign and tumorsamples in a blinded manner for both extent and intensity ofimmunoreactivity. Extent of immunostaining on one section was defined asthe epithelial area that showed positive δ-catenin immunoreactivity andwas scored semi-quantitatively as follows: 0, absent; 1+, <25%; 2+,25-50%; 3+, 51-75%; 4+, >75%. Intensity of immunostaining was scoredsemi-quantitatively as follows: 0, negative; 1+, weak; 2+, moderate; 3+,strong.

Two pathologists from two different groups assigned Gleason scores forthe TMA independently. When the TMA slides were studied for δ-cateninimmunoreactivity, one analysis was performed with the aid of automateddigital reading and an immunoscore was obtained as: extentscore×intensity score (range, 0 to 12). In another analysis, manualreading was applied, and the immunoscores for the staining intensity andstaining extent were assigned separately. For tumors with both samples(two representative 0.6 mm areas as described above) present, theimmunoscore was the mean of the two scores for the separately assessedsamples. They were analyzed semi-quantitatively by light microscopyequipped with MetaMorph imaging software (Universal Imaging Corp. WestChester, Pa.).

Data and Statistical Analyses.

To examine the relationship among δ-catenin, E-cadherin and p120^(ctn),the serial sections were photographed and the distribution andexpression of these proteins were analyzed. Statistical analysis wasperformed using MS Excel and SigmaPlot 5.0 (SPSS Science, Chicago,Ill.). Student t-tests were conducted and p-value was assigned. Thesignificance level was set at 0.05.

Results

δ-Catenin is overexpressed in prostate cancer cells. Previous studiesindicated that δ-catenin mRNA is expressed in tumor cell lines (Lu etal., supra 1999; Lu, Q. et al., J Neurosci Res. 67 (5): 618-624, 2002),and is overexpressed in prostate cancer in comparison to benign prostatehyperplasia (BPH) (Burger, M. J. et al., Int J Cancer 100(2): 228-237,2002). To determine if δ-catenin protein expression is increased inprostate cancer cells, we first compared human marrow stromal cells HS-5and non-cancerous human prostate epithelial cells PZ-HPV-7 with CWR-R1,a cell line derived from a recurrent CWR22 human prostate tumorxenograft (Pretlow, T. G. et al., J Natl Cancer Inst. 85 (5): 394-398,1993; Wainstein, M. A. et al., Cancer Res. 54 (23): 6049-6052, 1994;Nagabhushan, M. et al., Cancer Res. 56 (13): 3042-3046, 1996; Gregory,C. W. et al., Cancer Res. 61(7): 2892-2898, 20010). Although WesternBlots showed that δ-catenin is expressed in all of the cell lines, itsexpression in CWR-R1 cells is significantly higher than in HS-5 andPZ-HPV-7 cells (data not shown). δ-Catenin immunoreactivity is authenticsince it co-migrates with δ-catenin in cortical neurons (data notshown), where δ-catenin expression has been well established (Lu et al.,1999; Lu et al., 2002; Jones, S. B. et al., Neuroscience 115 (4):1009-1021, 2002). δ-Catenin expression in CWR-R1 cells has also beenconfirmed by the use of four independent δ-catenin antibodies: mAbD30,which recognizes amino acids 85-194; rAb62, which recognizes amino acidresidues 434-530; rAb64, raised against amino acid residues 828-1022;and rAb25, an antibody that recognizes amino acid residues 1201-1225(data not shown). To determine if the enhanced level of δ-cateninprotein in cancer cells detected by Western Blot might be due to anincreased mRNA expression of δ-catenin gene, we applied RT-PCR and foundthat CWR-R1 cells displayed increased amounts of δ-catenin mRNA whencompared with PZ-HPV-7 cells (data not shown). These resultsdemonstrated that δ-catenin protein is overexpressed in prostate cancercells in vitro. In addition, an enhanced mRNA expression is at leastpartially responsible for the overexpression of δ-catenin.

δ-Catenin expression is increased in human prostatic adenocarcinomas.

In the only study to previously examine changes in δ-catenin expressionin cancerous tissues, Burger et al (Id) compared δ-catenin mRNAexpression using real-time PCR in 16 prostate tumor and 11 BPH tissuesamples. These investigators reported that levels of δ-catenin mRNAexpression in all tumor samples examined were higher than in any of theBPH samples. However, no correlation between δ-catenin mRNA expressionand tumor progression was observed (Id.). To extend this study of theassociation between δ-catenin expression and the progression of humanprostate cancer, an immunohistochemical analysis of the δ-cateninprotein expression in human prostatic adenocarcinomas was conductedusing Tissue MicroArray (TMA).

We have performed immunohistochemistry on TMA samples that contained 90cases of prostatic adenocarcinomas and 90 benign prostatic tissuesamples (data not shown). Two pathologists from two groups independentlyassigned Gleason scores to the tumor specimens. One pathologistdetermined the Gleason scores without prior knowledge of patientinformation. In one analysis, an immunoscore was obtained as: extentscore×intensity score (range, 0 to 12, see Material and Method). Anestimated 85% of the prostate cancer specimens showed enhancedimmunoreactivity when compared to the benign prostatic tissue samplesfrom the peripheral zones of the glands (Table I). The mean±s.e. of theimmunoscore for benign samples was 2.52±0.04. For tumor samples, themean±s.e. of the immunoscore was 7.58±0.05 (FIG. 8, Panel A). In 65 of90 cases, both benign and tumors can be compared and assessed. Theimmunoscore was increased in tumor versus benign in 55 of 65 cases (or85%); whereas the immunoscore of cancer and benign samples was equal in6 out of 65 (or 9%) cases, and the immunoscore was higher in benignversus tumor in only 4 out of 65 (or 6%) cases (Table I). The mean tumorscores appeared to increase with prognostically significant increasedGleason scores. In this analysis, we observed an immunoscore of 6.24 inthe tumors with Gleason score 5.6; an immunoscore of 7.69 in the tumorswith Gleason score 7; an immunoscore of 8.75 in tumors with Gleasonscore>8 (FIG. 8, Panel B).

In an independent experiment in which protein lysates from 8 cases ofhigh-grade prostate tumors were compared to 8 cases of benign prostatetissues sampled from the peripheral zone of the glands, δ-catenin showedan increased immunoreactivity in 5 cases or ˜63% of all specimens (datanot shown). Semi-quantitative analysis showed that δ-cateninimmunoreactivity in prostate cancer lysates increased approximately 80%when compared to that in benign samples.

When prostate gland images were analyzed, anti-δ-catenin immunostainingwas very weak in benign tissues (data not shown). In the prostaticadenocarcinomas with low tumor grade, δ-catenin staining was localizedto the secretory glandular epithelial cells (data not shown). The numberof δ-catenin-positive cells expanded in prostate cancer samples withhigh tumor grade (data not shown).

These results together demonstrated that δ-catenin expression isincreased in primary human prostatic adenocarcinomas.

Increased Expression of δ-Catenin is Associated with the Downregulationof E-Cadherin and p120^(ctn) in Human Prostatic Adenocarcinomas.

In the second analysis in which consecutive sections were immunostainedwith anti-δ-catenin, anti-E-cadherin and anti-p120^(ctn), extent scoreand intensity score were determined separately to see if the resultsmight be different from that obtained in the first analysis describedabove. Here, 92% of the prostate cancer samples showed strong (46 out of72 cases) or moderate (20 out of 72 cases) staining (immunoscore equalor above 2) for δ-catenin, only 6 out of 72 (or 8%) of the specimens waseither negative or with immunoscore below 2 (Table II, Cancer).Interestingly, while 49 out of 65 cases (or 75%) of benign specimensshowed an immunoscore of below 2, 6 out of 65 (or 9%) of benign samplesshowed an immunoscore equal to 2, and 10 out of 65 (or 15%) of benignspecimens showed an immunoscore above 2. This data indicates that asmall percentage of cases will be falsely judged positive for prostatecancer if benign specimens are evaluated using δ-catenin immunoscore asthe sole criteria.

When anti-E-cadherin immunoreactivity was analyzed, 96% of benignspecimens showed strong (64 out of 71 cases) or moderate (4 out of 71cases) staining (immunoscores equal or greater than 2), only 3 out of 71cases (or 4%) of the benign specimens showed an weak immunoreactivity(less than 2), indicating an excellent correlation between strongE-cadherin expression and benign prostate tissues (Table II). However,anti-E-cadherin immunoreactivity was strong (39 out of 89 cases) ormoderate (20 out 89 cases) in 66% of the prostate cancer cases, withonly 30 out of 89 cases (or 34%) showed an immunoscore lower than 2.These observations showed that relying on E-cadherin immunoreactivityalone could have very high probability of missing prostate tumors,especially in the low Gleason score tumors. It should be noted thatthese data were derived from the average of total prostate cancer cases.In the most aggressive cases of prostatic adenocarcinomas (Gleasonscore>8), over 71% (or 10 out of 14 cases) of the specimens showed asharply reduced immunoscores below 2, consistent with the literaturethat E-cadherin is down regulated in the more advanced prostaticadenocarcinomas.

In benign specimens, anti-p120^(ctn) showed an overwhelmingly strongimmunoreactivity of above immunoscore 2 (71 out of 71 cases or 100%).However, 62 out 88 cases (or 70%) of the prostatic adenocarcinomasretained fairly strong anti-p120^(ctn) immunoreactivity of above 2, withonly 21% of the cases showing immunoscore below 2 (Table II). Thisindicates that additional biomarkers is necessary to compensate forp120^(ctn) for detecting prostate cancer, particularly in the lowGleason score tumors. Similar to anti-E-cadherin immunostaining,anti-p120^(ctn) staining was dramatically reduced in the more aggressivecases of prostatic adenocarcinomas (Gleason score>8), consistent withthe notion that both E-cadherin and p120^(ctn) are tumor suppressors.

Using δ-Catenin, E-Cadherin and p120^(ctn) as Biomarker Cluster Improvesthe Probability of Predicting or Excluding Prostate Cancer.

We further analyzed the data set to determine whether E-cadherin andp120^(ctn) immunoreactivity can be used to correct the false negativeresults associated with the 8% of the prostate cancer cases that showedweak anti-δ-catenin immunoreactivity. Our hypothesis was that from these6 cases where δ-catenin immunoscore was weak, perhaps the δ-cateninepitopes were not preserved appropriately during tissue processing, orδ-catenin level in some patients was inherently low. If this was thecase, employing anti-E-cadherin or anti-p120^(ctn) may circumvent theproblem of misdiagnosis. Indeed, in these 6 cases that showed weakδ-catenin immunoreactivity, anti-E-cadherin staining of the consecutivesections was able to correctly predict prostate cancer in 4 cases (or67%), while anti-p120^(ctn) immunoreactivity can compensate for 83% ofthe time (or 5 out of 6 cases) (Table III). The probability to correctthese 6 false negative cases stands at 83% when both data fromanti-E-cadherin and anti-p120^(ctn) staining were used (Table IV). Thiswould result in only 1 case out of total 72 prostatic adenocarcinomas(or 1.4%) that showed a false negative in predicting prostate cancerwhen examining 6-catenin, E-cadherin and p120^(ctn) as a biomarkercluster.

As shown above, anti-E-cadherin and anti-p120^(ctn) showed strongimmunostaining in both benign and the majority of prostaticadenocarcinomas, particularly in the low Gleason score specimens.Therefore, although the down regulation of these two proteins wasclosely associated with the high Gleason score prostaticadenocarcinomas, it would not be safe to rely on these markers topredict early stage prostate cancer. Table II showed that, in 39 out of89 (or 44%) of the prostate cancer specimens, E-cadherinimmunoreactivity remained strong. When these 39 cases were evaluated foranti-δ-catenin and anti-p120^(ctn) staining, 24 out of 39 cases (or 62%)showed strong immunoreactvity for δ-catenin and 5 out of 39 cases (or13%) showed reduced immunostaining of p120^(ctn) (Table III). Becausethe correction by δ-catenin and p120^(ctn) immunoreactivity happened inthe overlapping cases, there was a total 62% of chances that theseprostate cancer cases will be discovered if all three biomarkers wereused in predicting prostatic adenocarcinomas (Table IV). In prostatecancer specimens, 62 out of 88 (or 70%) of the specimens showed stronganti-p120^(ctn) immunoreactivity, while only 18 out of 88 (or 21%) ofthe cases displayed a clearly reduced anti-p120^(ctn) staining intensity(Table II). The majority of these 18% cases came from the high Gleasonscore prostatic adenocarcinomas. When anti-δ-catenin immunoreactivity inthese 62 cases was analyzed, 31 cases (or 58%) showed enhancedimmunoscores, whereas 29 out of 62 cases (or 47%) showed reducedE-cadherin signal, consistent with the presence of prostaticadenocarcinomas in these cases (Table III). Therefore, here thecombination of anti-δ-catenin and E-cadherin immunostaining cancorrectly detect 49 out of 62 (or 79%) prostate cancer cases thatotherwise would be judged falsely negative using anti-p120^(ctn)immunostaining as the only criteria (Table IV).

Similarly, using δ-catenin, E-cadherin and p120^(ctn) as biomarkercluster can reduce false positive outcomes and increase the probabilityin excluding prostate cancer misdiagnosis in benign populations. Forexample, 7 cases of benign tissues showed moderate or reducedanti-E-cadherin immunoreactvity (Table II). Anti-δ-cateninimmunoreactivity was able to correct 4 out of 7 cases (or 57%). 10 cases(or 15%) of benign tissues showed strong anti-δ-cateninimmunoreactivity, while 6 cases (or 9%) of benign samples showedmoderate anti-δ-catenin immunoreactivity. This result suggested that 16out of 65 benign cases might be misinterpreted as having prostatecancer. When these cases were examined using anti-E-cadherin as amarker, 13 of them (or 81%) showed strong immunoreactivity. Although allbenign specimens (71/71 or 100%) showed strong anti-p120^(ctn)immunoreactivity (Table II), p120^(ctn) immunoreactivity is also strongin many cases of early stage prostatic adenocarcinomas. Therefore,relying on p120^(ctn) immunoreactivity alone may lead to false negativeresults. Since most of the prostate cancer cases that showed falselystrong E-cadherin and p120^(ctn) staining did not show the same falselyweak δ-catenin staining (Table III and Table IV), the non-overlappingnature of the false positive and false negative cases suggest that it ispossible to exclude many of the false positive outcomes if these threebiomarkers are used for analysis.

Increased δ-Catenin Expression is Associated with the Down Regulation ofE-Cadherin and p120^(ctn) During the Progression of ProstaticAdenocarcinomas.

Interestingly, δ-catenin immunoreactivity increased significantly inhigh-grade prostate intraepithelial neoplasia (PIN), the most likelypre-invasive stage of prostatic adenocarcinomas. With increasing Gleasonscore, δ-catenin immunoreactivity continued to increase the intensityand extent until the prostatic adenocarcinoma reach Gleason score 10. Atthis stage, while the extent of δ-catenin staining remained the same,the intensity of δ-catenin staining decreased somewhat (FIG. 9, Panels Aand B). The E-cadherin and p120^(ctn) immunoreactivities, on the otherhand, showed a reciprocal trend in expression when compared withδ-catenin immunoreactivity (FIG. 9, Panels C-F). E-cadherin expressiondecreased significantly in PIN, but remained at the same level as in PINin the tumors with Gleason scores 6 and 8. Then in Gleason 10 tumors,E-cadherin immunoreactivity reduced dramatically, reflecting thedispersion and the invasiveness of the tumor cells. The intensity ofp120^(ctn) expression did not go down significantly in PIN and Gleason 6tumors. Even in the Gleason 8 tumors, the intensity and the extent ofp120^(ctn) immunoreactivity remained quite strong. However, in theGleason 10 tumors, both the intensity and extent of p120^(ctn)immunoreactivity reduced sharply (FIG. 9, Panels E and F).

Increased δ-Catenin Expression is Accompanied by the Redistribution ofE-Cadherin and p120^(ctn) in the Same Tumor Clusters of ProstaticAdenocarcinomas.

One advantage of analyzing the serial sections of a single case is thatall three cell-cell junction biomarkers in the same tumor cell clusterscan be compared. In benign prostate glandular epithelial cells,δ-catenin immunoreactivity was quite weak (data not shown).Anti-E-cadherin immunoreactivities were strong and were concentrated atthe cell-cell contact area, consistent with its localization to theadherens junction (data not shown). There was very littleanti-E-cadherin immunoreactivity in the cytoplasm. Similarly, p120^(ctn)immunoreactivity was clearly localized at the cell-cell junction (datanot shown). In the low-grade prostatic adenocarcinomas (Gleason score6), δ-catenin immunoreactivity was generally increased, and can be moreevident in some cells than others in the same tumor clusters (data notshown). In some of these tumor clusters, the relationship among theimmunoreactivities of δ-catenin, E-cadherin and p120^(ctn) can bedirectly compared. For example, δ-catenin immunoreactivity wasjunctional in the central part of the tumor cluster. However, in theperipheral cell layer of the tumor cluster, not only the intensity ofδ-catenin immunoreactivity was higher, but also most of the signalbecame non-junctional. When E-cadherin and p120^(ctn) immunoreactivitywere analyzed in the same tumor clusters, they showed strong junctionalstaining corresponding to the center of the cluster (data not shown),but became more cytoplasmic at the periphery of the tumor cluster (notshown). Nevertheless, the intensity and the extent of anti-E-cadherinand anti-p120^(ctn) immunostaining did not reduce significantly in manyof the low-grade prostatic adenocarcinomas. In the prostaticadenocarcinomas with high Gleason score of 10, cell-cell junctions wereseverely disrupted. Here, anti-δ-catenin immunoreactvity showed intensecytoplasmic staining (not shown). Most of the E-cadherin and p120^(ctn)immunoreactivity at the cell-cell junction was either lost orredistributed to the cytoplasm (not shown).

Forced δ-Catenin Overexpression in CWR-R1 Derived from Human ProstateCancer Xenograft Induces the Reduction of E-Cadherin and p120^(ctn) atthe Cell-Cell Junction.

Because δ-catenin binds to the same JMD region on E-cadherin asp120^(ctn), an increased δ-catenin expression may compete withp120^(ctn) for E-cadherin binding. As shown above, increased δ-cateninexpression in prostatic adenocarcinomas indeed corresponded to the lossof E-cadherin and p120^(ctn) at the cell-cell junction and theirconcomitant increases in cytoplasmic immunoreactivities (data notshown). To further examine the hypothesis that an increased δ-cateninexpression can affect the E-cadherin and p120^(ctn) at the cell-celljunction, we overexpressed δ-catenin ectopically into prostateepithelial cells in culture.

CWR-R1 cells (Gregory et al., Cancer Res. 61(7): 2892-2898, 2001) werederived from a recurrent human prostate cancer xenograft (Pretlow etal., supra 1993; Wainstein, M. A. et al., Cancer Res. 54 (23):6049-6052, 1994). These cells grow in clusters in culture and displayedan enhanced δ-catenin immunoreactivity when compared with PZ-HPV-7, anon-tumorigenic human prostate epithelial cell line. The level ofδ-catenin expression in CWR-R1 was moderate when compared with that inneurons (not shown). This moderate level of δ-catenin expression wasalso demonstrated in culture by immunofluorescent light microscopy. Inthe cell clusters, δ-catenin was localized at the cell-cell junction(not shown). In the same cell clusters, E-cadherin immunostainingcorresponded mainly to the cell-cell contact area (not shown) and atleast partially co-localized with δ-catenin at the cell-cell junction(not shown). When pEGFP-δ-catenin was overexpressed in CWR-R1 cells bytransfection, its localization at the cell-cell junction was clearlydemonstrated (not shown), along with some cytoplasmic immunoreactivity.Here, the purple line demarcated the untransfected cells in the field.When E-cadherin immunoreactivity was examined, the untransfected cellsshowed the same cell-cell junction staining. But the cells transfectedwith δ-catenin showed a reduced E-cadherin immunoreactivity (not shown).When the merged images were analyzed, no co-localization of E-cadherinand δ-catenin at the cell-cell junction area could be demonstrated.

We also examined the p120^(ctn) immunoreactivity and distribution in theCWR-R1 cells transfected with pEGFP-δ-catenin. Untransfected cellsshowed p120^(ctn) localization at the cell-cell junction (data notshown). However, cells transfected with δ-catenin showed the reducedp120^(ctn) immunoreactivity (data not shown). In addition, p120^(ctn)staining at the cell-cell junction was very weak, which was also shownby the lack of merged yellow staining at the cell-cell contact area(data not shown). As an additional control, CWR-R1 cells weretransfected with EGFP vector alone, which uniformly distributed in thecytoplasm (data not shown). The untransfected cells were highlightedwith the red lines (not shown). Here, anti-p120^(ctn) immunoreactivitywas localized to the cell-cell junction, whether in transfected oruntransfected cells (not shown). The merged images showed strong redsignals in most of the cells (not shown).

TABLE I Paired analysis of δ-catenin immunoscores in benign versusprostate tumor sections. Immunoscore Cancer > Benign Cancer = BenignCancer < Benign Number of 55/65 6/65 4/65 Cases (85%) (9%) (6%) *Out of90 cases, total 65 benign sections and 65 prostate tumor sections can beused for direct, paired comparison

TABLE II Comparison of δ-catenin, E-cadherin and p120^(ctn)immunoscores* in predicting prostate cancer. Immunoscore Benign CancerMarkers Strong Moderate Weak Strong Moderate Weak δ- 10/65 6/65 49/65 46/72 20/72  6/72 catenin (15%) (9%) (75%)  (64%) (28%)  (8%) E-cad-64/71 4/71 3/71 39/89 20/89 30/89 herin (90%) (6%)  (4%) (44%) (22%)(34%) p120^(ctn) 71/71 0/71 0/71 62/88  8/88 18/88 (100%)  (0%) ( )%)(70%)  (9%) (21%) *Total 65 benign cases and 72 prostatic adenocarcinomaspecimens stained with anti-δ-catenin, 71 benign cases and 89 prostaticadenocarcinoma specimens stained with anti-E-cadherin, and 71 benigncases and 88 prostatic adenocarcinoma samples stained with p120^(ctn)can be used for direct comparison

TABLE III The comparison of the ability of δ-catenin, E-cadherin andp120^(ctn) immunoscores in correcting each other's false negativeoutcomes in predicting prostate cancer. Cases of false negative by onebiomarker δ-catenin E-cadherin p120^(ctn) immunoreactivity: correctioncorrection correction δ-catenin 0/6 4/6 5/6 (n = 6)  (0%) (67%) (83%)E-cadherin 24/39  0/39  5/39 (n = 39) (62%)  (0%) (13%) p120^(ctn) 31/6229/62  0/62 (n = 62) (50%) (47%)  (0%)

TABLE IV Probability of successful prediction of prostate cancer usingδ-catenin- E-cadherin-p120^(ctn) immunoscore as a biomarker cluster.Cases of false negative by one biomarker Successful correction usingimmunoreactivity: δ-catenin/E-cadherin/p120^(ctn) δ-catenin  5/6 (83%)(n = 6) E-cadherin 24/39 (62%) (n = 39) p120^(ctn) 49/62 (79%) (n = 62)

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method for detecting breast cancer in asubject comprising the steps of: obtaining a sample from said subject;contacting said sample with anti-delta-catenin antibodies selected fromthe group consisting of mAbD30, rAb62, rAb64 and rAb25 to form ananti-delta-catenin antigen complex; and detecting the presence ofanti-delta-catenin antigen complex, wherein the presence ofantigen-antibody complex indicates the presence of cancer, wherein saidcancer is breast cancer, and wherein said delta-catenin is also known asthe adherens-junction linked arm (ALARM) protein.
 2. The method of claim1, wherein said subject is a human.
 3. The method of claim 1, whereinsaid subject is female.
 4. The method of claim 1, wherein the sample isselected from the group consisting of blood, urine, breast tissue, andbreast tissue fine needle aspirate fluid samples.
 5. The method of claim1, wherein the sample is a tissue sample, cell sample or stroma sample.6. The method of claim 5, wherein an intervening culturing step isperformed between the time the cell sample is obtained from the subjectand an immunoassay is carried out on the cell sample.
 7. The method ofclaim 1 further comprising contacting said sample with delta-cateninspecific primers to form a delta-catenin PCR reaction product; anddetecting the presence of delta-catenin PCR reaction product, whereinthe presence of delta-catenin PCR product indicates the presence ofcancer.
 8. The method of claim 1, wherein the anti-delta-cateninantibody is mAbD30.
 9. The method of claim 1, wherein theanti-delta-catenin antibody is rAb62.
 10. The method of claim 1, whereinthe anti-delta-catenin antibody is rAb64.
 11. The method of claim 1,wherein the anti-delta-catenin antibody is rAb25.
 12. A method fordetecting breast cancer in a subject comprising: contacting a cellsample or a tissue sample from a breast of the subject with ananti-delta-catenin antibody selected from the group consisting ofmAbD30, rAb62, rAb64 and rAb25 to form an anti-delta-catenin antigencomplex; and detecting the presence of anti-delta-catenin antigencomplex, wherein the presence of antigen-antibody complex indicates thepresence of cancer, wherein said cancer is breast cancer, and whereinsaid delta-catenin is also known as the adherens-junction linked arm(ALARM) protein.
 13. The method of claim 12, wherein said subject isfemale.
 14. The method of claim 12, wherein a cell sample is contactedwith the anti-delta-catenin antibody to form an anti-delta-cateninantigen complex.
 15. The method of claim 12 further comprising measuringthe distribution of anti-delta-catenin antigen complex in a cell-celljunction and the cytoplasm.